CARACTERÍSTICAS DEL DEPARTAMENTO DE CAAZAPA

Various morphometric methods, such as manual quantification, of stained tissues or cells have been conventionally used in routine laboratory analyses. When studying the testis, immunohistochemistry and immunofluorescence staining are considered helpful in the identification of specific cell types. However, using morphometric techniques in a heterogeneous organ like the testis is laborious and time-consuming.

Thus, using flow cytometry technology to study testicular populations coupled to DNA staining and/or specific antibodies has improved the screening of testicular cell composition ^137–140. Unlike previously reported protocols, we proposed a flow-cytometry-based protocol that allows quick assessment of rodent testis phenotype in one working day.

5.1.1 Protocol development and optimization

The principal goal of this study is to develop an accurate and fast flow-cytometry-based method for the analysis of testicular cell populations. As we wanted to obtain

information on different types of cell populations, signaling cascades, and molecular pathways, we opted to develop a protocol suitable for fluorescent intracellular antibody detection. While developing our method, we used rat testis ontogenesis as a model (5, 10, 16, and 24 days old and adult rats) as the testicular composition and maturation at different time points are well characterized ^211,212.

Testicular single-cell suspensions are prepared in four steps: mincing of the tissue, enzymatic/mechanical dissociation, fixation, and permeabilization (I; Figure 1). The enzymatic and mechanical step was optimized step by step to obtain representative testicular cell population ratios (I; Figure 1) that mimic the natural cell dynamics during rodent testicular development. To avoid the destruction of fragile pachytene spermatocytes, the maximum number of mechanical pipetting steps during enzymatic incubation was set to five. Fixation and permeabilization of the cell suspension are considered critical steps to use intracellular antibodies, for which we used 4% PFA and cold methanol, respectively. This protocol enables the storage of testicular single-cell suspensions for long periods of time at −20℃. If needed, these suspensions can also be delivered to other laboratories for further staining and analysis.

To distinguish the heterogeneous cell populations in our single-cell suspension, we used propidium iodide or fxCycle as a DNA stain. Flow cytometry analysis showed that we recovered representative amounts of testicular cell populations. In rats that are 5–16 days old, the testicular cell suspension was found to consist of 2C cells in the S-phase of the cell cycle and 4C populations. Notably, the ratio of 2C (SCs, spermatogonia) to 4C (primary spermatocytes) populations was found to change when 1C (spermatids) cells were observed in the testes of 24-day-old rats.

By the time the animal reaches adulthood, the 1C population becomes predominant in comparison to the 2C and 4C populations (I; Figure 3). According to these results, the cell number rates in different cell populations obtained by our optimized protocol were found to be similar to those in previous morphometric studies ^211,216.

5.1.2 Intracellular antibody staining

To confirm the presence of representative populations and to gather more information about the somatic/germ cell dynamics, we optimized the fluorescence intracellular staining technique to the obtained single-cell suspension. We used vimentin antibody (expressed in SCs and Leydig cells) to monitor the somatic cells of the testis and phosphorylated γH2AX antibody to detect germ cells. After intracellular antibody staining, the single-cell suspension was counterstained with a DNA stain and analyzed using flow cytometry.

The expression of vimentin was found to mimic the proliferation dynamics of somatic cells during rat testicular development ^211,213 (I; Figure 4). Before

spermatogenesis, the composition of the rat testis consisted mostly of SCs and spermatogonia. As expected, the rat testicular cell population at the ages of 5 and 10 days was found to consist mostly of vimentin-positive cells. At the age of 16 days, the proportion of vimentin-positive cells starts to decrease as the proliferation of SCs gradually ceases to form the BTB, and spermatogonia begin to proliferate. From the age of 24 days, when the first haploid cells emerge, to the adult age, the relative abundance of vimentin-positive cells decreases as the seminiferous tubules consist mostly of germ cells at different differentiation steps (I; Figure 4). This protocol is considered to be helpful in recovering a representative amount of somatic cells of young and adult testes.

In the testis, phosphorylated γH2AX histone protein is associated with both DNA double-stranded breaks and sex vesicle formation. This protein is expressed in germ cells from differentiated spermatogonia (2C) to pachytene spermatocytes (4C) and in steps 11 to 14 in RSs (1C) ^214,215. From our results, at the age of 16 days, most of the testicular cells were negative for γH2AX, because somatic cells were undergoing proliferation, and undifferentiated spermatogonia were present (I; Figure 5). As expected, most of the 4C populations were positive for γH2AX staining in the adult testis (I; Figure 5). These results prove that this antibody can be useful in the detection of specific subsets of germ cells in rodents.

Despite the broad availability of antibodies on the market, validation of immunofluorescence staining for flow cytometry analysis is challenging because many antibodies do not share the same staining protocols. Our aim was to use multiple antibodies at once; however, some of the antibodies (PLZF, SOX9, SMA, KI67, and protamine 2) did not perform as expected. This phenomenon may be related to the fixation and permeabilization steps that alter the target epitopes.

Additionally, interspecies differences may have limited the performance of these antibodies, restricting the availability and use of suitable antibodies among rodents.

Despite the advantages of our method and the large number of cells that can be analyzed using a flow cytometer, this method cannot replace the golden-standard histological analysis. However, our approach can provide much more targeted information to understand and complement histological data.

5.1.3 Application of the flow-cytometry-based method

Since the publication of the flow-cytometry-based method, it has been adapted to be used with different amounts of rat and mouse testicular tissues (Table 4).

Table 4. Application of the flow cytometry protocol in additional projects.

Studies Specie Analyzed tissue Staining

Eggert A., Cisneros-Montalvo S., Anandan S., Musilli S., Stukenborg J.B., Adamsson A., Nurmio M., Toppari J.

2019. The effects of PFOA (Perfluorooctanoic acid) on fetal and adult rat testis. Reprod Toxicol. 11;90:68-76.

Mouse 20 pieces of 2

Hakkarainen, J., Zhang F.P., Jokela H., Mayerhofer A., Behr R., Cisneros-Montalvo S., Nurmio M., Toppari J., Ohlsson C., Kotaja N., Sipila P., and Poutanen M. 2018.

Hydroxysteroid (17beta) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility. Faseb J. 32:3229-3241.

Mouse 10mg of testis tissue per sample

DNA staining

Reda, A., Albalushi H., Montalvo S.C., Nurmio M., Sahin Z., Hou M., Geijsen N., Toppari J., Soder O., and Stukenborg J.B. 2017. Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures. Ann.Biomed.Eng.

Rotgers, E., Nurmio M., Pietilä E., Cisneros-Montalvo S., and Toppari J. 2015. E2F1 controls germ cell apoptosis during the first wave of spermatogenesis.

Andrology. 3:1000-1014.

Mouse 10mg of testis tissue per sample

DNA staining

5.1.4 Update and future perspectives (I)

Notably, after we published our protocol, we succeeded in downscaling it, allowing us to analyze only a few milligrams of testis tissues ²¹⁷ and even pieces of the seminiferous tubules ²¹⁸ (Table 4.). Our protocol can be used for quick screening of testicular phenotypes of transgenic animals and testicular effects of in vivo drug exposure, and it can also be used to study the cell dynamics of in vitro cultures of seminiferous tubule pieces. Recently, we have added the phospho-histone H3 (Ser10) antibody to our antibody panel to analyze mitotic and meiotic progression during testicular development.

In the future, further optimization of a larger panel of specific antibodies would provide additional information on somatic and germ cell dynamics and signaling pathways during testicular development and spermatogenesis, which would have significant potential in several clinical applications, such as the characterization, diagnosis, and monitoring of disorders of human male reproduction.

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